Lee P C, Park I Y, Kim M S, Kim S C
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea.
J Biotechnol. 1998 Jul 16;62(3):177-85. doi: 10.1016/s0168-1656(98)00059-5.
Effect of the replication mode of a plasmid on the stability of tandemly multimerized endoxylanase genes and a gene dose-dependent expression of the endoxylanase were studied in Bacillus subtilis. The structural genes encoding an endoxylanase, carrying its original promoter and ribosomal binding sequence, were tandemly multimerized and cloned into the Escherichia coli-B. subtilis shuttle plasmid, pJH27 delta 88 or pMTL500e, which has a rolling circle-replicon or a theta (theta)-replicon in B. subtilis, respectively. The cloned dimers in pJH27 delta 88, which has a rolling circle-replicon, spontaneously rearranged to monomers in B. subtilis DB104, whereas those in pMTL500e, having a theta (theta)-replicon, were stably maintained. Expression level of the endoxylanase was proportional to the gene dosage in multimers. The endoxylanase activity in the supernatant increased from 80 U ml-1 with pMTL-1x containing a monomer of the gene to 165 U ml-1 with pMTL-4x containing a tetramer. These results indicate that high level expression of the endoxylanase gene can be obtained by tandemly multimerizing the genes in a plasmid with a theta (theta)-replicon.
在枯草芽孢杆菌中研究了质粒复制模式对串联多聚化内切木聚糖酶基因稳定性的影响以及内切木聚糖酶基因剂量依赖性表达。编码内切木聚糖酶的结构基因，携带其原始启动子和核糖体结合序列，被串联多聚化并克隆到大肠杆菌 - 枯草芽孢杆菌穿梭质粒pJH27 delta 88或pMTL500e中，它们在枯草芽孢杆菌中分别具有滚环复制子或θ（theta）复制子。在具有滚环复制子的pJH27 delta 88中克隆的二聚体在枯草芽孢杆菌DB104中自发重排为单体，而在具有θ（theta）复制子的pMTL500e中的那些则稳定维持。内切木聚糖酶的表达水平与多聚体中的基因剂量成正比。上清液中的内切木聚糖酶活性从含有该基因单体的pMTL - 1x的80 U ml-1增加到含有四聚体的pMTL - 4x的165 U ml-1。这些结果表明，通过在具有θ（theta）复制子的质粒中串联多聚化基因可以获得内切木聚糖酶基因的高水平表达。
